The invention relates to a new technology of extracting tetrodotoxin from the tissues of organisms, including but not limited to puffer fish. The invention can increase the yield of tetrodotoxin from puffer fish at least three times over that of methods of prior art.
Tetrodotoxin is a nonprotein neurotoxin that is found in multiple diverse animal species, including puffer fish, goby fish, newt, frogs and the blue-ringed octopus.
Tetrodotoxin can be obtained from a variety of organisms. The puffer fish, especially its ovaries, is the most well-known source of tetrodotoxin. Tetrodotoxin has also been isolated from species of frog (J. W. Daly et al., Toxicon 32:279 (1996)) and in goby fish (T. Noguchi et al., Toxicon 11:305 (1973)). It has been hypothesized that colonizing bacteria may be responsible for tetrodotoxin biosynthesis in marine organisms (J. W. Daly, 1996).
The known methods for extraction and purification of tetrodotoxin optimally provide 1 to 2 grams of tetrodotoxin from 100 kg of puffer fish ovaries.
The chemical name of tetrodotoxin and other related data are shown below:
Chemical name: Octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol
Molecular formula: C12H17N3O8 
Molecular weight: 319.27
Structure: 
Pure tetrodotoxin is a colorless crystalline powder. It darkens above 220xc2x0 C. without decomposition. There are several hydrophilic hydroxyl groups in tetrodotoxin, making it insoluble in organic solvents. Its molecular skeleton is similar with the cage structure of adamantane, making hydration very difficult, thus it is just slightly soluble in water. Because there is a guanidine perhydroquinazoline group in the molecule, and guanidine group is strongly alkaline, tetrodotoxin is soluble in water solutions of acids. Because tetrodotoxin also has the structure of inner ester, and water solutions of strong acid will make it decompose, the only way to keep it stable in solution is to dissolve it in a solution of weak organic acid. The particularity of C-4 can be easily seen from the molecular structure of tetrodotoxin. C-4 is in the ortho-position of the nitrogen atom with OH group at equatorial position and H atom at axial position. Therefore, the chemical and biological activities of the hydroxyl group on C-4 are significant. If H+ is present in the solution, the oxygen atom from the hydroxyl group of C-4 will combine with it, and produces positively charged structure B from structure A. Structure B losses a H20 molecule and result in structure C with positively charged C-4. 
Structure C may interact with H2O in the solution. The H2O can either attack the position where the original H2O molecule is eliminated so that structure E is derived, or attack the opposite position where the H2O molecule is eliminated so that structure D is derived. If a H2O molecule is removed from structure E, the original tetrodotoxin structure A is produced. Structure D will lead to structure F after a H2O is removed. The difference between structure F and A is that the positions of H and OH are exchanged. The H on C-4 of structure A is axial and OH is equatorial, while in structure F the H on C-4 is equatorial and OH is axial. 
Tetrodotoxin structure A is called xe2x80x9ctetrodotoxinxe2x80x9d, which is the predominant content of tetrodotoxin (TTX) obtained from puffer fish in the nature. Tetrodotoxin structure F is usually called 4-epi tetrodotoxin. Because the hydroxyl group on C-4 is close to the one on C-9 in 4-epi tetrodotoxin, a H2O molecule is easily removed under the interaction of H+, resulting in an analog of tetrodotoxin containing ether bond, which is called 4-epi anhydrotetrodotoxin. The chemical properties of these three xe2x80x9ctetrodotoxinxe2x80x9d molecules are only slightly different. But they have significant differences in terms of biological activities. For example, the toxicity of tetrodotoxin is 4500 mouse units/milligram; 4-epi tetrodotoxin, 710 mouse units/milligram; 4-epi anhydrotetrodotoxin, only 92 mouse units/milligram (Toxicon, 23 271--276(1985)).
The importance of C-4 hydroxyl group is also manifested in that its toxicity will be substantially reduced when it is replaced by other groups, such as H, CH3 or CH3COxe2x80x94. Hence, theoretically, efforts should be made to keep the hydroxyl group on C-4 equatorial during the extraction of tetrodotoxin. So it is very important to select the right extraction devices and materials, pH and temperature of the solution, and time for the extraction process. The toxin extracted from puffer fish is a mixture that consists of more than 10 analogs, predominant among which is tetrodotoxin, accounting for 70% to 80% of the mass of the extract. Three other major analogs are tetrodonic acid, 4-epi tetrodotoxin and 4-epi anhydrotetrodotoxin, which are only slightly different in chemical properties but significantly different in biological activities. For example, the toxicity of tetrodotoxin is 4500 mouse units/milligram; 4-epi tetrodotoxin, only 710 mouse units/milligram; 4-epi anhydrotetrodotoxin, dramatically decreased to 92 mouse units/milligram (Toxicon, 23, 271-276(1985)).
Two approaches, synthesis and extraction from biological sources, have been used to obtain tetrodotoxin. In 1972 Y. Kishi et al succeeded in completely synthesizing tetrodotoxin. At the beginning of the 20th century, Tahara in Japan started extracting tetrodotoxin (J. Pharm. Soc. Japan 29. 587(1909), Biochem.Z.30 255(1911)). He used lead acetate and aqueous ammonia to precipitate toxin and lead together, then removed the lead by treatment with hydrogen sulfide. Tahara further added methanol and diethyl ether to the filtrate after removal of the lead, and finally precipitated the toxin. Tahara measured the toxicity of the crude toxin to be 4.1xcex3, (The mean lethal dose to 1 gram of mouse is MLD 4.1xcex3/g, abbreviated as 4.1xcex3y), and named the toxin as xe2x80x9ctetrodotoxinxe2x80x9d, as the toxin is called today. In the decades following, many extraction methods were developed; representative methods are:
In 1950, Yokoo successfully obtained crystalline toxin of MLD 0.01xcex3 (J. Chem. Soc. Japan 71, 590 (1950)). He obtained the ovaries from the puffer fish caught near Shimomi, Japan at the end of January, lixiviated them in water and steamed the product dry. The result was a dry substance of MLD 40xcex3. Next, lead acetate and ammonia were used to precipitate toxin and lead together, then the impurities were removed with phospho-tungstic acid and mercury picrate; sugars were removed with phenyl hydrazine. Mercury picrate was used again to treat the precipitate, followed by treatment with picrolonic acid, methanol, and picrolonic acid so that crystalline toxin of MLD 0.8xcex3 was obtained. But out of 20 kg of ovaries, Yokoo obtained only 13 mg crystalline toxin.
In 1951, Nagai (Fukuoka Ishi 45, 1 (1954)) used ion exchange resin (Amberlite IRC-50) to adsorb toxin, eluting with hydrochloric acid, then treating the eluate with Amberlite IR-4B to remove hydrochloric acid. After concentration, anhydrous alcohol was used to extract toxin. 2.5 mg toxin crystals of MLD 0.008xcex3 were finally obtained from 20 kg of the puffer fish ovaries.
In 1952, Tsuda and Kawamura used circular filter paper chromatography (K. Tsuda et al, J. Pharm. Soc. Japan 72, 187, 771(1952)) and obtained toxin of MLD=10 xcexcg/kg. Later they developed a method for mass production by active charcoal column chromatography (Tsuda, Kagaku no Ryouiki, supplement, 80, 9 (1967)), which was capable of processing 1000 kg of ovaries and obtained 10 grams toxin with MLD=10 xcexcg/kg. Meanwhile, Woodward (R. B. Boodward, Pure Appl. Chem. 9, 49-74 (1964)) achieved similar results.
In 1964, Goto Toshio et al (Goto Toshio and Takahashi et al., J. Chem. Soc. Japan 85, 508 (1964)) simplified the technological process by using ion exchange and active charcoal adsorption (See FIG. 1). They obtained 1-2 grams so-called crude toxin from 100 kg of ovaries.
After 1980, some methods were reported from time to time but they generally followed T. Goto""s method and failed to increase yield.
This invention makes significant improvements with respect to the yield-restraining factors that have been discovered by the inventors after years of research on the advantages and deficiencies of existing extraction technologies. The yield of tetrodotoxin by this invention is at least three times that previously reported in the literature.
The extraction of tetrodotoxin of this invention comprises five steps as follows:
Step 1
Grind the tissues into small pieces, soak with an amount of water equal to 1.5 times by weight of the tissues and an amount of a weak organic acid, typically a carboxylic acid, preferably acetic acid, equal to 0.05%-1%, preferably 0.1%-0.3%, by weight of the tissue for several hours, then stir and filter quickly to obtain a lixiviated solution. Repeat this step 3-4 times in order to extract as much toxin as possible.
Step 2
Heat the lixiviated solution to 70-95xc2x0 C. to coagulate and remove soluble proteins (xe2x80x9cscleroproteinxe2x80x9d).
Step 3
Adjust the pH of the lixiviated solution obtained in step 1 to 6.0xcx9c7.5 using an aqueous solution of a weak base, then put the solution through a weakly acidic cation ion-exchange resin to enrich tetrodotoxin. Elute the bound tetrodotoxin with a weak acid.
Step 4
Adjust the pH of the obtained tetrodotoxin solution in step 3 to 8 to 9 for a period of 2-4 hours, during which put the solution through a column filled with active charcoal and diatomaceous silica so as to remove inorganic salts and a fraction of the alkaline amino acids. Then wash the column with de-ionized water first, then with acidic ethanol solution, in order to elute as much toxin adsorbed as possible.
Step 5
Purify and crystallize tetrodotoxin by concentrating the solution obtained in step 4 under vacuum, then adjusting to an alkaline pH. Vacuum dry the obtained tetrodotoxin crystals, typically about 24 hours until the weight of the crystals becomes constant.